PROJECT SUMMARY Sudden Cardiac Death (SCD) is a major health problem in the United States and globally, accounting for 15%-20% of all deaths. Reduction of sodium current (INa) through the main cardiac voltage-gated sodium channel (Nav1.5) has been identified as a contributor to arrhythmias leading to SCD in many acquired and inherited cardiomyopathies. Two post-translational modifications (PTMs) in the cytoplasmic III-IV linking domain of Nav1.5, acetylation at K1479 and phosphorylation at S1503, have been found to decrease INa by reducing cell surface abundance of Nav1.5. Notably, both of these PTMs are reversed by increased intracellular NAD+ levels, providing a therapeutic strategy to increase INa. Preliminary studies found that Nicotinamide Riboside (NR), a NAD+ precursor, increases INa in vitro. However, it remains unknown how these Nav1.5 PTMs modulated by NAD+ and Nicotinamide Riboside can coordinate channel localization and influence cardiac electrophysiology in vivo. An additional PTM, ubiquination, is an established regulator in the internalization and trafficking of Nav1.5 and other sodium channels. Recent studies suggest ubiquination may be facilitated by other PTMs, a process known as ?PTM crosstalk?. The objective of my application is to determine if and how these PTMs on Nav1.5 regulate channel localization and to determine if these processes may be manipulated to affect arrhythmic risk in vivo. The overall hypothesis is that modulation of PTMs by Nicotinamide Riboside, an NAD+ precursor, disrupts crosstalk with ubiquination-endocytic processes, thereby increasing cell surface localization of Nav1.5 and modifying the arrhythmic risk. To test this hypothesis, two mouse models of Nav1.5 dysfunction will be utilized, in addition to cellular approaches. These studies will test the hypothesis through two specific aims: Aim 1: Determine the mechanism(s) driving Nav1.5 surface localization in response to increasing NAD+ content by examining post-translational modifications and endocytic processes in vitro. Aim 2: Determine if Nicotinamide Riboside supplementation can modify cardiac electrical activity in mouse models of Nav1.5 dysfunction.